Understanding
the thermodynamic properties of complex DNA nanostructures,
including rationally designed two- and three-dimensional (2D and 3D,
respectively) DNA origami, facilitates more accurate spatiotemporal
control and effective functionalization of the structures by other
elements. In this work fluorescein and tetramethylrhodamine (TAMRA),
a Förster resonance energy transfer (FRET) dye pair, were incorporated
into selected staples within various 2D and 3D DNA origami structures.
We monitored the temperature-dependent changes in FRET efficiency
that occurred as the dye-labeled structures were annealed and melted
and subsequently extracted information about the associative and dissociative
behavior of the origami. In particular, we examined the effects of
local and long-range structural defects (omitted staple strands) on
the thermal stability of common DNA origami structures. The results
revealed a significant decrease in thermal stability of the structures
in the vicinity of the defects, in contrast to the negligible long-range
effects that were observed. Furthermore, we probed the global assembly
and disassembly processes by comparing the thermal behavior of the
FRET pair at several different positions. We demonstrated that the
staple strands located in different areas of the structure all exhibit
highly cooperative hybridization but have distinguishable melting
temperatures depending on their positions. This work underscores the
importance of understanding fundamental aspects of the self-assembly
of DNA nanostructures and can be used to guide the design of more
complicated DNA nanostructures, to optimize annealing protocol and
manipulate functionalized DNA nanostructures